Biocatalytic microcapsules for catalyzing gas conversion

ABSTRACT

According to one embodiment, a microcapsule for selective catalysis of gases, the microcapsule comprising: a polymeric shell permeable to one or more target gases; and at least one biocatalyst disposed in an interior of the polymeric shell. In more embodiments, methods of forming such microcapsules include: emulsifying at least one biocatalyst in a polymer precursor mixture; emulsifying the polymer precursor mixture in an aqueous carrier solution; crosslinking one or more polymer precursors of the polymer precursor mixture to form a plurality of microcapsules each independently comprising: a polymeric shell permeable to one or more target gases; and at least one biocatalyst disposed in an interior of the polymeric shell. In further embodiments, corresponding methods of using the inventive microcapsules for catalyzing one or more target gases using include: exposing a plurality of the biocatalytic microcapsules to the one or more target gases.

The United States Government has rights in this invention pursuant toContract No. DE-AC52-07NA27344 between the United States Department ofEnergy and Lawrence Livermore National Security, LLC for the operationof Lawrence Livermore National Laboratory.

FIELD OF THE INVENTION

The present invention relates to bioreactors, and more particularly tobiocatalytic microcapsules providing improved surface area and masstransport to facilitate conversion of target gases using biocatalyst(s)in the biocatalytic microcapsules.

BACKGROUND

Most chemical reactions of interest for clean energy are routinelycarried out in nature. These reactions include the conversion ofsunlight to chemical energy, the transfer of carbon dioxide into and outof solution, the selective oxidation of hydrocarbons (including methaneto methanol), the formation of C—C bonds (including methane toethylene), and the formation and dissolution of Si—O bonds (includingenhanced mineral weathering). Conventional industrial approaches tocatalyze these reactions are either inefficient or have yet to bedeveloped.

Certain enzymes have been identified that carry out each of theaforementioned reactions. Unfortunately, industrial biocatalysis isprimarily limited to the synthesis of low-volume, high-value products,such as pharmaceuticals, due to narrow operating parameters required topreserve biocatalyst activity.

Conventional gas conversion involves enzyme-catalyzed reactionstypically carried out in fermenters, which are closed, stirred, tankreactors configured to use bubbled gases for mass transfer. FIG. 1 ,illustrates a conventional stirred tank reactor 100, which may include amotor 102, an input/feed tube 104, a cooling jacket 106, one or morebaffles 108, an agitator 110, one or more gas spargers 112, and anaqueous medium 114. Gas exchange in the stirred tank reactor 100 may beachieved by bubbling from the sparger(s) 112 at the bottom of theaqueous medium 114 and gas collection above said aqueous medium 114.Care must be taken to maintain a narrow set of conditions in suchstirred tank reactors to favor the desired metabolic pathways anddiscourage competing pathways and competing organisms. Moreover, stirredtank reactors are energy inefficient, require batch processing, sufferfrom loss of catalytic activity due to enzyme inactivation, and exhibitslow rates of throughput due to low catalyst loading and limitedmass-transfer resulting from low solubility of the target gases in theliquid medium and relatively low surface area of the bubbled gases.Although surface area may be improved with increased stirring/agitation,this exacerbates the energy inefficiency of the stirred tank reactor andis an impractical solution to improving gas conversion efficiency.

To allow reuse of enzymes in stirred-tank reactors, and to improvestability in reactor conditions, enzymes may be immobilized on inert,artificial materials. As shown in FIG. 2 , one conventional approach isto immobilize enzymes 202 on the surface of an inert material 204. Otherconventional approaches may involve immobilizing enzymes on the surfaceof accessible pores of inert materials. However, such conventionalenzyme immobilization techniques also suffer from lower volumetriccatalyst densities, low throughput rates, and do not have routes forefficient gas delivery or product removal. Accordingly, it would beadvantageous to provide systems and techniques enabling the re-use ofcatalytic components while also providing high surface area and masstransport to improve the catalyst density, throughput, and efficiency ofgas conversion.

In addition, certain applications in which gas conversion is important,e.g. preservation of organic or gas-sensitive materials such as food,medicine, etc. may prohibit the use of a liquid medium to facilitatemass transport. Accordingly, it would be a further advantage to providesystems and techniques enabling efficient gas conversion using drycompositions capable of effective mass transport and catalysis topreserve the sensitive material.

SUMMARY

According to one embodiment, a microcapsule for selective catalysis ofgases, the microcapsule comprising: a polymeric shell permeable to oneor more target gases; and at least one biocatalyst disposed in aninterior of the polymeric shell.

In accordance with another embodiment, a method of forming microcapsulesfor selective catalysis of gases includes: emulsifying at least onebiocatalyst in a polymer precursor mixture; emulsifying the polymerprecursor mixture in an aqueous carrier solution; crosslinking one ormore polymer precursors of the polymer precursor mixture to form aplurality of microcapsules each independently comprising: a polymericshell permeable to one or more target gases; and at least onebiocatalyst disposed in an interior of the polymeric shell.

In further embodiments, a method for catalyzing one or more target gasesusing biocatalytic microcapsules includes: exposing a plurality of thebiocatalytic microcapsules to the one or more target gases.

Other aspects and embodiments of the present invention will becomeapparent from the following detailed description, which, when taken inconjunction with the drawings, illustrate by way of example theprinciples of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

For a fuller understanding of the nature and advantages of the presentinvention, reference should be made to the following detaileddescription read in conjunction with the accompanying drawings.

FIG. 1 . is a schematic representation of a conventional stirred tankreactor, according to the prior art.

FIG. 2 is a schematic representation of enzymes immobilized on anexterior surface of an inert material, according to the prior art.

FIG. 3 is a schematic representation of enzymatic reactive componentsembedded within a polymeric network, according to one embodiment.

FIG. 4 is a process flow illustrating a method for embedding enzymaticreactive components within a two phase (AB) polymer network, accordingto one embodiment.

FIG. 5 is a process flow illustrating a method for embedding enzymaticreactive components within a two phase (AB) polymer network, accordingto another embodiment.

FIG. 6 is schematic representation of a bioreactor comprising a hollowtube network/lattice configured to optimize mass transfer, according toone embodiment.

FIG. 7 is a flowchart of a method for forming a bioreactor via 3Dprinting, according to one embodiment.

FIG. 8 illustrates various photographs of PEG-pMMO 3D structuresformed/patterned according to a projection microstereolithography (PμSL)process, according to e embodiments.

FIG. 9 is a process flow illustrating a method for forming apolyethylene glycol diacrylate (PEGDA) hydrogel comprising particulatemethane monooxygenasae (pMMO), according to one embodiment.

FIG. 10A is a plot illustrating pMMO retention by weight in a PEGDAhydrogel as a function of the volume percentage of PEGDA present duringpolymerization, where 150 μg of pMMO is initially included within thePEGDA hydrogel.

FIG. 10B is a plot illustrating pMMO activity in a PEGDA hydrogel as afunction of the volume percentage of PEGDA present duringpolymerization, where 150 μg of pMMO is initially included within thePEGDA hydrogel.

FIG. 10C is a plot illustrating pMMO retention by weight in a PEGDAhydrogel as a function of the amount of pMMO (μg) included duringpolymerization.

FIG. 10D is a plot illustrating the activity of PEGDA-pMMO and a pMMOcontrol as a function of the amount of pMMO (μg) included during theactivity assay.

FIG. 11A is a plot illustrating the activity of the PEGDA-pMMO hydrogelafter reusing said hydrogel over multiple cycles.

FIG. 11B is a plot illustrating the amount of methanol (nmoles) producedper mg of pMMO for both as-isolated membrane bound pMMO and PEGDA-pMMOover twenty cycles of methane activity assay.

FIG. 12A is a schematic representation of a continuous flow-throughPEGDA-pMMO hydrogel bioreactor, according to one embodiment.

FIG. 12B is a plot illustrating the amount of methanol (nmole) producedper mg of pMMO in the PEGDA-pMMO hydrogel bioreactor of FIG. 12A.

FIG. 13 is a plot illustrating the dependence of PEGDA-pMMO activity onsurface area to volume ratio for a PEGDA-pMMO hydrogel bioreactor.

FIG. 14A is a simplified schematic of a biocatalytic microcapsule inaccordance with one embodiment of the present disclosures.

FIG. 14B is a photographic representation of a plurality of biocatalyticmicrocapsules, according to another embodiment.

FIG. 15 depicts a simplified schematic of a microcapsule assemblyapparatus according to one approach.

FIG. 16 is a flowchart of a method for forming biocatalyticmicrocapsules for selective catalysis of target gases, according to oneembodiment.

FIG. 17 is a flowchart of a method for catalyzing one or more targetgases using biocatalytic microcapsules, according to one embodiment.

DETAILED DESCRIPTION

The following description is made for the purpose of illustrating thegeneral principles of the present invention and is not meant to limitthe inventive concepts claimed herein. Further, particular featuresdescribed herein can be used in combination with other describedfeatures in each of the various possible combinations and permutations.

Unless otherwise specifically defined herein, all terms are to be giventheir broadest possible interpretation including meanings implied fromthe specification as well as meanings understood by those skilled in theart and/or as defined in dictionaries, treatises, etc.

it must also be noted that, as used in the specification and theappended claims, the singular forms “a,” “an” and “the” include pluralreferents unless otherwise specified.

As also used herein, the term “about” when combined with a value refersto plus and minus 10% of the reference value. For example, a length ofabout 100 nm refers to a length of 100 nm+10 nm.

As further used herein, the term “fluid” may refer to a liquid or a gas.

As discussed previously, enzymes have been identified that catalyzevirtually all of the reactions relevant to clean energy, such asselective transformations among carbon fuels, gas to liquids technology,storage of solar energy, exchange of CO₂, formation and dissolution ofsilicates, and neutralization of wastes. However, industrial enzymebiocatalysis is currently limited to low-volume, high-value productssuch as pharmaceuticals due to the narrow operating parameters requiredto preserve biocatalyst activity; slow rates of throughput due to lowcatalyst loading; limited mass transfer; and susceptibility tocontamination and poisoning. These limitations require that manybiocatalysis processes are carried out in single phase, aqueous mediasuch as that provided in stirred tank reactors. However, stirred tankreactors are energy inefficient, require batch processing, and have poormass transfer characteristics. While techniques have emerged to improvethe stability and allow reuse of enzymes in stirred tank reactors, suchtechniques involve immobilizing the enzymes solely on the exteriorsurface(s) of an inert material or on the exterior surface(s) of thepores of an inert material. Unfortunately, these conventionalimmobilization techniques still fail to rectify the slow throughputrates and limited mass transfer associated with current biocatalysisprocesses.

To overcome the aforementioned drawbacks, embodiments disclosed hereinare directed to a novel class of bioreactor that includes a membranecomprising one or more types of reactive enzymes and/orenzyme-containing cell fragments embedded within, and throughout thedepth of, a multicomponent polymer network. In various approaches, thismulticomponent polymer network may comprise two or more polymer types,or a mixture of a polymer and inorganic material. Preferably, themembrane comprises permeable, multi-component polymers that serve asboth a mechanical support for the embedded enzymes, as well asfunctional materials configured to perform one or more additionalfunctions of the bioreactor, such as: efficiently distributing reactantsand removing products; exposing the embedded enzymes to highconcentrations of reactants; separating reactants and products; forminghigh surface area structures for exposing the embedded enzymes toreactants; supplying electrons in hybrid enzyme-electrochemicalreactions; consolidating enzymes with co-enzymes in nanoscale subdomainsfor chained reactions, etc. In additional approaches, this membrane maybe molded into shapes/features/structures (e.g., hollow fibers,micro-capsules, hollow tube lattices, spiral wound sheets, etc.) tooptimize the bioreactor geometry for mass transfer, product removal, andcontinuous processing.

The novel class of bioreactor disclosed herein may be especially suitedto catalyze reactions that occur at phase boundaries, e.g., gas toliquid, liquid to gas, polar to non-polar, non-aqueous to aqueous, etc.Accordingly, the novel class of bioreactors disclosed herein may beuseful for reactions in clean energy applications that involve agas-phase reactant or product (e.g., methane to methanol conversion, CO₂absorption, oxidation reactions with O₂, reduction reactions with H₂ ormethane, CO₂ conversion to synthetic fuel, etc.), as well as reactionsin the chemical and pharmaceutical industries that involve treatment ofnon-polar organic compounds with polar reactants (or vice versa).

The following description discloses several general, specific, andpreferred embodiments relating to biocatalytic microcapsules providingimproved surface area and mass transport to facilitate conversion oftarget gases using biocatalyst(s) in the biocatalytic microcapsules.

According to one general embodiment, a microcapsule for selectivecatalysis of gases, the microcapsule comprising: a polymeric shellpermeable to one or more target gases; and at least one biocatalystdisposed in an interior of the polymeric shell.

In accordance with another general embodiment, a method of formingmicrocapsules for selective catalysis of gases includes: emulsifying atleast one biocatalyst in a polymer precursor mixture; emulsifying thepolymer precursor mixture in an aqueous carrier solution; crosslinkingone or more polymer precursors of the polymer precursor mixture to forma plurality of microcapsules each independently comprising: a polymericshell permeable to one or more target gases; and at least onebiocatalyst disposed in an interior of the polymeric shell.

In further general embodiments, a method for catalyzing one or moretarget gases using biocatalytic microcapsules includes: exposing aplurality of the biocatalytic microcapsules to the one or more targetgases.

Block Polymer/Copolymer Embodiments

Referring now to FIG. 3 , a membrane 300 particularly suitable for usein a bioreactor is shown according to one embodiment. As an option, themembrane 300 may be implemented in conjunction with features from anyother embodiment listed herein, such as those described with referenceto the other FIGS. Of course, the membrane 300 and others presentedherein may be used in various applications and/or in permutations whichmay or may not be specifically described in the illustrative embodimentsliked herein. For instance, the membrane 300 may be used in any desiredenvironment and/or include more or less features, layers, etc. thanthose specifically described in FIG. 3 .

As shown in FIG. 3 , the membrane 300 includes a plurality of enzymaticreactive components 302 embedded within a polymer network 304. Invarious approaches, the enzymatic reactive components 302 may compriseabout 1% to 80% of the mass of the polymer network 304. The enzymaticreactive components 302 may be configured to catalyze any of thereactions described herein, and in particular reactions that take placeat phase boundaries (e.g., gas to liquid, liquid to gas, polar tonon-polar, non-aqueous to aqueous, etc.).

In some approaches, the plurality of enzymatic reactive components 302may comprise one or more of: isolated enzymes, trans-cell-membraneenzymes, cell-membrane-bound enzymes, liposomes coupled to/comprising anenzyme, etc. Stated another way, each enzymatic reactive component 302may individually be selected from the group selected from: an isolatedenzyme, a trans-cell-membrane enzyme, a cell-membrane-bound enzyme, anda liposome coupled to/comprising an enzyme. Suitable enzymatic reactivecomponents 302 may include, but are not limited to, formatedehydrogenase, carbonic anhydrase, cytochrome p450, hydrogenase,particulate methane monooxygenasae (pMMO), photosynthetic complexes,etc. Moreover, while each of the enzymatic reactive components 302 maybe the same (e.g., comprise the same structure and/or composition) inparticular approaches; other approaches may require at least two of theenzymatic reactive components 302 to be different (e.g., have adifferent structure and/or composition) from one another.

In approaches where at least one of the enzymatic reactive components302 includes a membrane-bound enzyme, said enzyme may be stabilizedprior to incorporation into the polymer network 304. For instance, inone stabilization approach, cell fragments comprising the enzyme ofinterest may be used, and directly incorporated into the polymer network304. In another stabilization approach, a lipopolymer may first beformed by linking a lipid to a polymer of interest. The lipid region ofthe polymer may spontaneously insert into the cell membrane, therebycreating a polymer functionalized liposome, which may be incorporated inthe polymer network 304. In yet another stabilization approach, theenzyme of interest may be coupled to and/or encapsulated into anano-lipo-protein particle (NLP), which may then be incorporated in thepolymer network 304.

The enzymatic reactive components 302 may be incorporated into thepolymeric network 304 via several methods including, but not limited to:attaching the enzymatic reactive components 302 to electrospun fibers ofa first polymer, and backfilling with a second polymer (see, e.g., themethod described in FIG. 4 ); directly incorporating the enzymaticreactive component 302 into a polymer or block-copolymer network beforeor after crosslinking the network (see, e.g., the method described inFIG. 5 ); and other suitable incorporation methods as would becomeapparent to one having skill in the art upon reading the presentdisclosure.

With continued reference to FIG. 3 , the polymeric network 304 mayinclude at least a two phase polymer network, e.g. a polymer networkcomprising two or more polymeric materials. This polymer network 304 maybe configured to serve as a mechanical support for the enzymaticreactive components 302 embedded therein, concentrate reactants, andremove products. In preferred approaches, the polymeric network 304 mayinclude nanometer scale domains of higher reactant permeability, as wellas nanometer scale domains of higher product permeability.

In particular approaches involving gas to liquid reactions, thepolymeric network may include nanometer scale domains of higher gaspermeability, such as silicon, as well as nanometer scale domains ofhigher product permeability, such as a polyethylene glycol (PEG) basedhydrogel. These domains of high gas permeability typically also havehigher gas solubility, increasing the local concentration of reactants(e.g., relative to the aqueous medium in a stirred tank reactor) andtherefore increase the turnover frequency of the enzymatic reactivecomponents 302; whereas, the domains of low gas permeability and highproduct permeability may efficiently remove the product and reduceproduct inhibition (thereby also increasing the turnover frequency andstability of the enzymatic reactive components 302) or serve tostabilize the enzymatic reactive components. In various approaches, thepermeability for the “higher gas permeability phase” may be greater than100 barrer.

In some approaches, the polymer network 304 may comprises a di-blockcopolymer network. In other approaches, the polymer network 304 mayinclude a tri-block copolymer network. Suitable polymers for thepolymeric network 304 may include silicone polymers,polydimethylsiloxane (PDMS), poly(2-methyl-2-oxazoline) (PMOXA),polyimide, PEG, polyethylene glycol diacrylate (PEGDA), poly(lacticacid) (PLA), polyvinyl alcohol (PVA), and other such polymers compatiblewith membrane proteins and block copolymer synthesis as would becomeapparent to one skilled in the art upon reading the present disclosure.In more approaches, each polymer in the polymeric network 304 may have amolecular weight ranging from about 500 Daltons to about 500kiloDaltons.

In other approaches, the polymeric network 304 may include a mixture ofat least one polymer material and at least one inorganic material.

In various approaches, a thickness, t₁, of the enzyme embedded polymernetwork 304 may be in a range from about 1 micrometer to about 2millimeters.

As indicated above, the membrane 300 may be configured to separate thereactants and products associated with a catalyzed reaction of interest.These reactants and products may be two different fluids, such asliquids and gasses, aqueous species and non-aqueous species, polarspecies and non-polar species, etc. In one exemplary approach where themembrane 300 may be configured to separate methane and oxygen frommethanol, the methane reactant concentration may be in a range fromabout 1 to about 100 mM, the oxygen reactant concentration may be in arange from about 1 to about 100 mL, and the methanol productconcentration range may be in a range from about 0.1 to about 1000 mM.

To further facilitate reactant-production separation, at least a portionof one surface of the membrane 300 may include an optional reactantpermeable polymer layer 306 coupled thereto, as shown in FIG. 3 . Inpreferred approaches, this reactant permeable polymer layer 306 may alsobe impermeable to products generated from the reactions catalyzed by theenzymatic reactive components 302. Suitable polymeric materials for thisreactant permeable polymer layer 306 may include, but are not limitedto, nanofiltration, reverse-osmosis, or chemically selective membranes,such as poly(ethylene imine), PVA, poly(ether ether ketone) (PEEK),cellulose acetate, or polypropylene (PP). In some approaches, athickness, t₂, of the reactant permeable polymer layer 306 may be in arange from about 0.1 to about 50 micrometers. This optional reactantpermeable polymer layer 306 may be particularly suited for approachesinvolving an organic polar reactant and an organic non-polar product andvice versa).

As also shown in FIG. 3 , at least a portion of one surface of themembrane 300 may include an optional product permeable polymer layer 308coupled thereto. This product permeable polymer layer 308 may preferablybe coupled to a surface of the membrane 300 opposite that on which thereactant permeable polymer layer 306 is coupled, thereby facilitatingentry of reactants (e.g., gaseous reactants) on one side of the membrane300, and removal of the reaction products (e.g., liquid reactionproducts) on the opposing side of the membrane 300. In more preferredapproaches, this product permeable polymer layer 308 may also beimpermeable to the reactants introduced into the enzyme embedded polymernetwork 304. Suitable polymeric materials for this product permeablepolymer layer 308 may include, but are not limited to, nanofiltration,reverse-osmosis, or chemically selective membranes, such aspoly(ethylene imine), PVA, poly(ether ether ketone) (PEEK), celluloseacetate, or polypropylene (PP). In some approaches, a thickness, t₃, ofthe product permeable polymer layer 308 may be in a range from about 0.1to about 50 micrometers.

In some approaches, one or more of the enzymatic reactive components 302may require a cofactor for function. Accordingly, cofactors may besupplied by co-localized enzymes in reactor domains of the polymernetwork 304 (not shown in FIG. 3 ), and/or be retained within a cofactorimpermeable layer coupled to a portion of the membrane 300 (not shown inFIG. 3 ).

In various approaches, a total thickness, t₄, of the membrane 300 may bein a range from about 10 to about 3100 micrometers.

In yet more approaches, the membrane 300 may be shaped into features,structures, configurations, etc. that provide a desired surface area tosupport efficient transport of reactants to, and products from, theenzymatic reactive components 302. For instance, the membrane 300 may beshaped into at least one of: a hollow fiber membrane, a micro-capsulemembrane, a hollow tube membrane, a spiral wound membrane, etc.

Referring now to FIG. 4 , a method 400 for embedding enzymatic reactivecomponents within a two phase (AB) polymer network is shown according toone embodiment. As an option, the present method 400 may be implementedin conjunction with features from any other embodiment listed herein,such as those described with reference to the other FIGS. Of course,this method 400 and others presented herein may be used to formstructures for a wide variety of devices and/or purposes, which may ormay not be related to the illustrative embodiments listed herein. Itshould be noted that the method 400 may include more or less steps thanthose described and/or illustrated in FIGS. 4 , according to variousembodiments. It should also be noted that that the method 500 may becarried out in any desired environment.

As shown in FIG. 4 , an enzymatic reactive component 402 is adsorbed toat least one portion of the exterior surface of polymer A 404, therebyforming enzyme-embedded polymer A 406. In preferred approaches, polymerA 404 may comprise one or more hydrophobic, reactant permeable (e.g.,gas permeable) polymeric materials configured to provide highconcentrations and fast transport of reactants. In further approaches,polymer A 404 may be a polymer nanofiber generated usingelectrospinning, extrusion, self-assembly, or other suitable techniqueas would become apparent to one skilled in the art upon reading thepresent disclosure. In additional approaches, such a polymer A nanofibermay be crosslinked to other polymer A nanofibers. In one exemplaryapproach, polymer A 404 comprises PDMS.

In various approaches, the enzymatic reactive component 402 may beselected from the group consisting of: an isolated enzyme, an enzymecomprising a cell fragment (e.g., a cell membrane or cell membranefragment), and a liposome comprising/coupled to an enzyme. In someapproaches, the enzymatic reactive component 402 may include at leastone of: formate dehydrogenase, carbonic anhydrase, cytochrome p450,hydrogenase, particulate urethane monooxygenasae (pMMO), photosyntheticcomplexes, etc.

In the non-limiting embodiment shown in FIG. 4 , a plurality ofenzymatic reactive components 402 may be adsorbed to one or moreportions of the exterior surface of polymer A 404. These enzymaticreactive components 402 may be adsorbed to at least the majority, ormore preferably about an entirety, of the exterior surface of polymer A404. The lipid bilayer vesicles of the enzymatic reactive components 402may spontaneously collapse on the exterior surface of polymer A 404,thereby forming a lipid-bilayer functionalized surface.

As further shown in FIG. 4 , the enzyme-embedded polymer A 406 may bemixed with polymer B 408 to create the two phase (AB) polymer monolith410 with the enzymatic reactive components 402 at the interface betweenthe two phases. In preferred approaches, polymer B 408 may comprise oneor more hydrophilic, product permeable polymeric materials configured toprovide transport of products, as well as stabilize the enzymaticreactive components 402. For instance, in one specific approach, polymerB 408 may be a hydrophobic polymer hydrogel.

While the resulting polymeric network shown in FIG. 4 includes twophases (i.e., polymer A and polymer B), it is important to note thatsaid polymeric network may include more than two phases in additionalapproaches.

Referring now to FIG. 5 , a method 500 for embedding enzyme reactivecomponents within a two phase (AB) polymer network is shown according toanother embodiment. As an option, the present method 500 may beimplemented in conjunction with features from any other embodimentlisted herein, such as those described with reference to the other FIGS.Of course, this method 500 and others presented herein may be used toform structures for a wide variety of devices and/or purposes, which mayor may not be related to the illustrative embodiments listed herein. Itshould be noted that the method 500 may include more or less steps thanthose described and/or illustrated in FIGS. 5 , according to variousembodiments. It should also be noted that that the method 500 may becarried out in any desired environment.

As shown in FIG. 5 , enzymatic reactive components 502 may be directlyincorporated in a block copolymer network 504 prior or aftercross-linking said network. As described herein, each enzymatic reactivecomponent 502 may be independently selected from an isolated enzyme, anenzyme comprising a cell fragment (e.g., a cell membrane or cellmembrane fragment), and a liposome comprising/coupled to an enzyme. Insome approaches, the enzymatic reactive component 502 may include atleast one of: formate dehydrogenase, carbonic anhydrase, cytochromep450, hydrogenase, particulate methane monooxygenasae (pMMO),photosynthetic complexes, etc.

As shown in the non-limiting embodiment of FIG. 5 , the block copolymernetwork 504 is a di-block copolymer network comprising two differentpolymers (polymer A 506 and polymer B 508). In preferred approaches,polymer A 506 may comprise one or more reactant permeable, hydrophobicpolymeric materials, whereas polymer B 508 may comprise one or moreproduct permeable, hydrophilic polymeric materials. It is againimportant to note that while the block copolymer network 504 shown inFIG. 5 includes two phases (i.e., polymer A 506 and polymer B 508), saidblock copolymer network may include more than two phases in otherapproaches.

In various approaches, the enzymatic reactive components 502 may beincorporated directly into the block copolymer network 504 usinglipopolymers (preferably di-block lipopolymers). Lipopolymers may begenerated by linking a lipid to a polymer of interest, such as PEG,creating PEG-lipid conjugates, such as PEG-phosphatidylethanolamie. Thelipid region of the polymer may spontaneously insert into the cellmembrane, thereby creating a polymer functionalized liposome.

Referring now to FIG. 6 , a bioreactor 600 comprising a network/latticeof three dimensional structures configured to optimize mass transfer isshown according to one embodiment. As an option, the bioreactor 600 maybe implemented in conjunction with features from any other embodimentlisted herein, such as those described with reference to the other FIGS.Of course, the bioreactor 600 and others presented herein may be used invarious applications and/or in permutations which may or may not bespecifically described in the illustrative embodiments listed herein.For instance, the bioreactor 600 may be used in any desired environmentand/or include more or less features, layers, etc. than thosespecifically described in FIG. 6 .

As noted above, the bioreactor 600 includes a network/lattice 602 ofthree dimensional structures. As particularly shown in FIG. 6 , thenetwork/lattice 602 includes multiple layers (e.g., 2, 3, 4, 5, 6, 7, ormore layers, etc.) of three-dimensional (3D) hollow tubes 604. It isimportant to note, however, that the hollow tube network/lattice 602 ofthe bioreactor 600, and others disclosed herein, may include one or morelayers of three-dimensional hollow tubes 604 in various approaches. Thehollow tubes 604 may preferably be oriented in the lattice such thattheir hollow interiors are perpendicular to a thickness direction of thelattice (e.g., perpendicular to the z axis shown in FIG. 6 ).

In some approaches, the bioreactor 600 may have a thickness (as measuredparallel to the z-axis in FIG. 6 ) in a range from about 1 to about 300cm, and a length (as measured in a direction parallel to the y-axis ofFIG. 6 ) and width (as measured in a direction parallel to the x-axis ofFIG. 6 ) scaled to the application, ranging from about 2 cm forlaboratory applications to 10 meters for industrial applications.

The walls of each hollow tube 604 may comprise a membrane material 606,such as the membrane material of FIG. 3 , configured to separatereactants (e.g., gaseous reactants) and products (e.g., hydrophilicproducts). Accordingly, the hollow tubes 604 form polymer microchannelsthrough which the hydrophilic reaction products may flow.

As particularly shown in FIG. 6 , the membrane material 606 of eachhollow tube 604 may comprise a plurality of enzymatic reactivecomponents 608 (e.g., isolated enzymes, membrane-bound enzymes,liposomes comprising/couple to an enzyme, etc.) embedded throughout apolymer network 610. The polymer network 610 may comprise reactantpermeable fibrils of a first polymer 612 that increase the localconcentration of reactants and enhance mass transfer throughout themembrane material 606. In some approaches, the enzymatic reactivecomponents 608 may be immobilized on the fibrils of the first polymer612. The polymer network 610 may also include at least another polymermaterial (e.g., a hydrogel matrix material) configured to hydrate theenzymatic reactive components 608 and provide a route for hydrophilicproduct removal. The membrane material 606 may also include an optionalreactant permeable (product impermeable) layer 614 coupled to one side(e.g., an exterior side) of the polymer network 610 and/or a productpermeable (reactant impermeable) layer 616 coupled to the opposite side(e.g., an interior side) of the polymer network 610. The optionalproduct permeable (reactant impermeable) layer 616 may also facilitateproduct removal and prevent coenzyme and/or cofactor diffusion into theliquid core that contains the desired products.

The thickness, t_(mem), of the membrane material 606 may be in a rangefrom about 10 to about 1000 micrometers. In some approaches, t_(m) maybe about 300 μm. Additionally, The thickness, t_(tube), of each hollowtube 604 may be in a range from about 10 micrometers to about 10millimeters. In various approaches, t_(tube) may be about 1 mm. In yetmore approaches, the length, l_(tube), of each hollow tube 604 may be ina range from about 5 centimeters to about 10 meters.

It is important to note that while the cross section of each hollow tube604, as taken perpendicular to the y-axis of FIG. 6 , is shown acircular, this need not be the case. For instance, in other approaches,each hollow tube 604 may have a cross sectional shape that iselliptical, rectangular, square, triangular, irregular shaped, etc.Moreover, in preferred approaches, each hollow tube 604 may have thesame cross sectional shape, materials, and/or dimensions; however, thisagain need not be case. For instance, in alternative approaches, atleast one of the hollow tubes 604 may have a cross sectional shape,materials, and/or dimensions that are different than that of another ofthe hollow tubes 604.

In one particular approach, one or more of the hollow tubes 604 in atleast one of the layers may differ from one or more hollow tubes 604 inat least another of the layers with respect to: cross sectional shape,and/or one or more membrane material(s), and/or one or more dimensions.In another particular approach, one or more of the hollow tubes 604 inat least one of the layers may differ from at least another hollow tube604 in the same layer with respect to: cross sectional shape, and/or oneor more membrane materials, and/or one or more dimensions.

In yet further approaches, the spacing between the hollow tubes 604 inat least one of the layers may be about uniform. In more approaches, thespacing between the hollow tubes 604 in at least one of the layers mayvary throughout the layer. For example, in one such approach, at leastone of the layers may have at least one area having an average spacing,s₁, between adjacent hollow tubes 604, and at least a second area havingan average spacing s₂, where s₁ and s₂ are different. In yet otherapproaches, the spacing between the hollow tubes 604 in at least one ofthe layers may differ from the spacing between the hollow tubes 604 ofat least another of the layers.

Referring now to method 7, an exemplary method 700 of forming abioreactor (such as those disclosed herein) is shown, according to oneembodiment. As an option, the present method 700 may be implemented inconjunction with features from any other embodiment listed herein, suchas those described with reference to the other FIGS. Of course, themethod 700 and others presented herein may be used in variousapplications and/or in permutations, which may or may not bespecifically described in the illustrative embodiments listed herein.Moreover, more or less operations than those shown in FIG. 7 may beincluded in method 700, according to various embodiments. Furthermore,while exemplary processing techniques are presented with respect to FIG.7 , other known processing techniques may be used for various steps.

As shown in FIG. 7 , the method 700 includes forming a membrane materialcomprising: a polymeric network configured to separate a first fluidfrom a second fluid, and a plurality of enzymatic reactive componentsembedded/incorporated within the polymeric network. See operation 702.

The enzymatic reactive components may comprise any of the enzymaticreactive components disclosed herein including, but not limited to,isolated enzymes, trans-cell-membrane enzymes, cell-membrane-boundenzymes, liposomes coupled to/comprising an enzyme, combinationsthereof, etc. Moreover, as discussed previously, the enzymatic reactivecomponents may be embedded/incorporated into the polymeric network viaseveral methods including, but not limited to: attaching the enzymaticreactive components to electrospun fibers of a first polymer, andbackfilling with a second polymer (see, e.g., the method described inFIG. 4 ); directly incorporating the enzymatic reactive component into apolymer or block-copolymer network before or after crosslinking thenetwork (see, e.g., the method described in FIG. 5 ); and other suitableincorporation methods as would become apparent to one having skill inthe art upon reading the present disclosure.

The polymeric network may include any of the materials, and/or be of thesame form, as any of the polymeric networks disclosed herein. Forinstance, this polymer network may be configured to serve as amechanical support for the enzymatic reactive components embeddedtherein, as well as include nanometer scale domains of higherpermeability to the first fluid and nanometer scale domains of higherpermeability to the second fluid. Moreover, in some approaches, thepolymeric network may include at least a two phase polymer network, e.g.a polymer network comprising two or more polymeric materials. In otherapproaches, the polymeric network may include a mixture of at least onepolymer material and at least one inorganic material.

As indicated above, the polymeric network may be configured to separatea first and second fluid associated with a reaction catalyzed by theenzymatic reactive components embedded therein. The first and secondfluids may be two different fluids, such as liquids and gasses, anaqueous species and a non-aqueous species, a polar species and anon-polar species, etc.

As also shown in FIG. 7 , the method 700 includes fabricating andpatterning one or more layers in the membrane material via a 3D printingprocess. See operation 704. In preferred approaches, the 3D printingprocess includes a_projection microstereolithography (PμSL) process asknown in the art. In various approaches, each layer in the membranematerial patterned/formed via the desired 3D printing process mayinclude a plurality of three dimensional structures (e.g., hollowfibers, micro-capsules, hollow tube lattices, spiral wound sheets, etc.)configured to optimize the bioreactor geometry (and surface area) formass transfer, reaction rate, product removal, continuous processing,etc. Photographs of several exemplary PEG-pMMO 3D structuresformed/patterned according to a PμSL process are shown in FIG. 8 .

As discussed in greater detail below, the novel bioreactors describedherein, such as described in FIG. 6 , may be particularly configured formethane activation with an energy efficiency from greater than or atleast equal to about 68%. In such an approaches, the enzymatic reactivecomponents embedded within the polymeric network may include pMMO tocovert methane reactants, CH₄, to methanol products, CH₃OH. Preferably,this engineered pMMO may exhibit a specific activity greater than about5 μm/(g·s) and/or a turnover frequency greater than about 10/s.Additionally, the amount of the engineered pMMO in such bioreactors maybe about 50 g per L of reactor volume.

In some approaches, the aforementioned engineered pMMO may require areducing agent for methane conversion. However, in other approaches, theengineered pMMO may not need such a reducing agent, or be configured toaccept electrons via direct electron transfer. For instance, as shown inTable 1, the methane conversion may proceed by: (1) using pMMOconfigured to use methane as a reducing agent (reaction 1); (2)supplying electrons directly to the pMMO (reaction 2); and (3) using H₂gas. Yet another reaction pathway may involve steam reformations shownin reaction 3.

TABLE 1 Energy Carbon Reaction Pathway efficiency efficiency Reaction 1:2CH₄ + O₂ → 2CH₃OH 80% 100% Reaction 2: CH₄ + O₂ + 2H⁺ + 2e⁻ →CH₃OH + >65% 100% H₂O Reaction 3: 4CH₄ + 3O₂ → 3CH₃OH + CO + 68% 75%2H₂O

EXPERIMENTS/EXAMPLES

The following experiments and examples pertain to various non-limitingembodiments of the bioreactors described herein. In particular, thefollowing experiments and examples are directed to bioreactorscomprising pMMO embedded in a polymeric network for the conversion ofmethane to methanol. It is important to note that the followingexperiments and examples are for illustrative purposes only and do notlimit the invention in anyway. It should also be understood thatvariations and modifications of these experiments and examples may bemade by those skilled in the art without departing from the spirit andscope of the invention.

Overview

Advances in oil and gas extraction techniques have made vast new storesof natural gas (composed primarily of methane) available for use.However, substantial quantities of methane are leaked, vented, or flaredduring these operations. Indeed, methane emissions contribute about ⅓ ofcurrent net global warming potential. Compared to other hydrocarbons,and especially compared to the oil that is co-produced inhydrofracturing operations, methane has a much lower market value due todifficulty in methane storage and transport, and because methane haslimited use as a transportation fuel.

Conversion of methane to methanol via conventional industrialtechnologies, such as steam reformation followed by the Fischer-Tropschprocess, operate at high temperature and pressure, require a largenumber of unit operations, and yield a range of products. Consequently,conventional industrial technologies have a low efficiency of methaneconversion to final products and can only operate economically at verylarge scales. There is thus a need in the art for a technology toefficiently convert methane to other hydrocarbons, and particularly toconvert “stranded” sources of methane and natural gas (sources that aresmall, temporary, or not close to a pipeline) to liquids for latercollection.

The only known true catalyst (industrial or biological) to convertmethane to methanol under ambient conditions with 100% selectivity isthe enzyme methane monooxygenase (MMO), found in methanotrophicbacteria, which converts methane to methanol according to the followingreaction:

Partial methane oxidation by MMO enzymes can be carried out using wholemethanotroph organisms, but this approach inevitably requires energy forupkeep and metabolism of the organisms, which reduces conversionefficiency. Moreover, biocatalysis using whole organisms is typicallycarried out in low-throughput unit operations, such as a stirred tankreactor.

One industrial-biological approach may therefor include separating theMMO enzyme from the host organism. Isolated enzymes may offer thepromise of highly controlled reactions at ambient conditions with higherconversion efficiency and greater flexibility of reactor and processdesign. MMOs have been identified in both soluble MMO (sMMO) andparticulate (pMMO) form. The use of pMMO has advantages for industrialapplications because pMMO comprises an estimated 80% of the proteins inthe cell membrane. Moreover, concentrating pMMO to a reasonable purityrequires only isolating the membrane fraction of the lysed cells usingcentrifugation.

Traditional methods of enzyme immobilization and exposure to reactantsare not sufficient to use pMMO effectively. These typical methodsinclude cross-linking enzymes or immobilizing them on a solid support sothat they can be separated from e products and carrying out batchreactions in the aqueous phase in a stirred tank reactor. As discussedpreviously, operation of a stirred tank reactor has several drawbacks,including low productivity, high operating costs, loss of catalyticactivity due to enzyme inactivation, and variability in the quality ofthe product. The stirred-tank reactor is also not the optimal design forgas to liquid reactions such as methane to methanol conversion, as itdoes not allow efficient delivery of reactant gases to enzymes ororganisms in the bulk solution. Gas delivery in stirred tank reactors isoften achieved by bubbling the gas through the liquid, but this approachsuffers from mass-transfer limitations. Furthermore, methane and oxygenare only sparingly soluble in aqueous solvents: 1.5 mM/atm and 1.3mM/atm respectively at 25° C. Reactant concentrations are necessarilysolubility-limited when the enzymes or organisms are dispersed in theaqueous phase.

Moreover, another reason as to why the pMMO enzyme is not amenable tostandard immobilization techniques designed for soluble proteins is dueto the fact that surfactant solubilization of isolated pMMO leads to apronounced reduction in activity. For example, high surface area porousinorganic supports have been extensively studied and implemented forimmobilizing soluble enzymes, and have been shown to enhance enzymestability while achieving high enzyme loading in nanometer scale pores.The majority of the surface area in mesoporous materials is accessibleonly to proteins significantly smaller than 50 nm, and would thereforebe inaccessible to the large (>100 nm), optically opaque vesicles andliposomes that comprise pMMO in crude membrane preparations.

Accordingly, the exemplary embodiments discussed in this experimentalsection are directed toward advances in biocatalytic processes forselective methane conversion. For instance, said exemplary embodimentsare particularly directed toward a biocatalytic material comprising pMMOembedded in polyethylene glycol diacrylate (PEGDA) hydrogel. Embeddingenzymes, such as pMMO that operate on gas phase reactants within thesolid, gas permeable polymer hydrogel allows tuning of the gassolubility, permeability, and surface area thereof. An additionaladvantage to immobilizing pMMO within the polymer hydrogel, rather thanon the surface of an impermeable support, is the potential to fullyembed pMMO throughout the depth of the polymer hydrogel for highloading. PEGDA was selected as the primary polymer substrate because ofits biocompatibility and flexibility for further development. PEGDA maybe physically or chemically combined with hydrophobic polymers inadditional approaches for enhanced gas solubility and transport invarious approaches. Moreover, the pMMO embedded PEGDA hydrogel isamenable to various forms of 3D-printing, which offers the ability torapidly prototype structures, tune micron to centimeter-scale materialarchitecture, and precisely tailor structures for the systemconfiguration and mass transfer, heat, and diffusion limitations.

Characterization of Block Copolymer Embodiments

a. pMMO Activity in PEG Hydrogel

Several methods for embedding pMMO in a PEGDA based polymer hydrogelwere explored to enable its use as a biocatalytic material which couldbe molded into controlled, predetermined structures with tunablepermeability and surface area for practical use. Initial efforts focusedon solubilizing the crude membrane preparations using surfactant so thatthe material could be incorporated homogeneously in the polymer. It wasdiscovered that any contact of the crude membrane preparations withsurfactant, including encapsulation in nanolipoprotein particles, led toa pronounced decrease in activity. However, mixing the crude membranefractions, either as prepared or extruded as liposomes directly with lowconcentrations of PEGDA 575 gave promising results. According theexperiments described in this section focused on optimizing the activityand protein retention of crude membrane preparations with PEGDA 575.

A schematic of the method used to fabricate the PEG-pMMO hydrogels isshown in FIG. 9 . The synthesis of the PEG-pMMO materials required onlymembrane bound pMMO, PEGDA macromer, photoinitiator (not shown), andultraviolet (UV) light. Photoinitiator concentrations higher than 0.5vol % in PEGDA decreased the pMMO activity, therefore the photoinitiatorconcentration was held constant at 0.5 vol %.

Membrane bound pMMO alone in each activity assay as a positive control.The measured activity of the membrane bound pMMO alone was highlyvariable from experiment to experiment, from about 75 to 200 nmol MeOHmg⁻¹ min⁻¹, while the optimized PEG-pMMO samples were less variable, ina range from 65 to 128 nmol MeOH mg⁻¹ min⁻¹. The measured activity forboth membrane bound pMMO alone and immobilized pMMO are similar to knownvalues for membrane bound pMMO with methane as a substrate: 25-130 nmolMeOH mg⁻¹ min⁻¹.

FIGS. 10A-10D shows the results from systematically increasing thevolume % of PEGDA in the solution prior to curing on protein retention(FIGS. 10A, 10C) and activity (FIGS. 10B, 10D). Mixing the pMMO solutionwith PEGDA at the appropriate vol % (10-80%), and UV curing resulted in50 μl solid PEG-pMMO hydrogels. As the PEGDA vol % was increased from10-80%, the overall stiffness of the material increased and the amountof residual liquid on the surface of the hydrogel decreased. A gradualincrease vas observed in the fraction of pMMO that was retained(0.4-0.75) when the PEGDA vol % was increased from 10-80% (FIG. 10A).

However, a dramatic decrease in pMMO activity was observed as the PEGDAvol % was increased (FIG. 10B) At 10% PEGDA, the pMMO activity wasapproximately 88+/−4 nmol MeOH min⁻¹mg⁻¹, which closely corresponded tothe activity of pMMO alone (96+/−15 nmol MeOH min⁻¹mg⁻¹) (FIG. 10B).This value dropped below 30 nmol MeOH min⁻¹mg⁻¹ when the PEGDA vol % wasgreater than 50% (FIG. 10B). The amount of pMMO retained in the hydrogelbefore and after the activity assay did not change, indicating that nopMMO leached out during the activity assay and the enzyme wasefficiently entrapped in the hydrogel. These combined findingsdemonstrate that one must consider both pMMO retention and activity whenidentifying the optimal PEGDA vol %. Since only a marginal increase inpMMO retention (0.4 vs 0.42) and a more significant decrease in pMMOactivity (88 vs 74 nmol MeOH min⁻¹mg⁻¹) was observed when the PEGDA vol% was increased from 10% to 20%, all remaining experiments wereperformed using 10 vol % PEGDA.

FIGS. 10C and 10D illustrate the effect of varying the concentration ofpMMO during hydrogel fabrication on pMMO retention and activity. Forthese experiments, the amount of pMMO used to generate the 50 μlPEG-pMMO hydrogel was varied between 50 μg and 550 μg. The fraction ofpMMO retained was the highest at the lowest pMMO concentration tested(50 μg-0.75 retained) and a dramatic decrease was observed when the pMMOwas increased to 150 μg (˜0.4 retained) (FIG. 10A). Further changes inthe total pMMO retained was not observed when the pMMO was increased upto 550 μg. To assess the effect of varying the pMMO concentrations inthe PEG-pMMO hydrogel on activity, PEG-pMMO hydrogels were prepared with50-550 μg of pMMO, which resulted in retention of 35-200 μg of pMMO inthe hydrogel, and the activity was measured. As shown in FIG. 10B, pMMOactivity in the hydrogel was similar to the activity of pMMO alone whenthe amount of pMMO retained was below 50 μg; however, there was agradual decrease in pMMO activity in the hydrogels as the pMMO levelswere increased from 50-200 μg, which was not observed in the pMMO alonesample (FIG. 10D).

Preserving the native activity of pMMO in the PEG hydrogel required abalance between pMMO loading and enzyme activity. Higher polymerconcentrations gave rise to higher pMMO loading and retention (FIG.10A). Increasing the polymer concentration also correlated withdiminished pMMO activity. This trend may be due to reduced polymerpermeability or enzyme degradation by acrylate groups and/or freeradicals at higher polymer concentrations. While it has been shown thatPEDGA concentration (and by correlation, crosslinking density) hasminimal effect on methane permeability in the gas phase, gaspermeability is affected by the hydration (swelling) of hydrogelmaterials. Thus, PEGDA concentration may impact methane permeability inswollen PEG-pMMO. Higher PEGDA concentrations also decrease the distancebetween crosslinks and the diffusion of aqueous solutes through thehydrogel. Therefore, higher PEGDA concentrations may limit diffusion ofthe NADH cofactor to the enzyme or diffusion of the methanol productfrom the active site. Additionally, photo-initiated cross-linkingreaction used to generate the cross-linked hydrogel results in thegeneration of free radicals, which can result in the oxidation of aminoacids in proteins and cleavage of peptide bonds. The optimized PEG-pMMOformulations described in the text were remarkable in that theypreserved physiological pMMO activity in a polymeric material; if ahigher protein or polymer content is required the above issues might bemanaged by changing the macromer length and/or curing chemistry, inorder to increase hydrogel mesh size (promoting diffusion) and reducethe number of radicals generated.

b. Reuse and Stability of PEG-pMMO Hydrogels

The development of fully active pMMO in a polymer material allowed thereuse of pMMO without painstaking centrifugation with each new set ofreactants. Measurements were made regarding the effects of reuse of thePEG-pMMO hydrogel on overall enzyme activity and methanol generationusing PEG-pMMO that was prepared with an initial pMMO amount of 150 μgand 1.0 vol % pMMO (FIGS. 11A-11B). In these experiments, the PEG-pMMOhydrogels were subjected to 20 cycles of 4 min exposures to methane. Thehydrogel was washed thoroughly between each cycle to ensure that noresidual methanol product remained in the hydrogel between cycles. Theprotein content in the reaction buffer for each cycle was measured toverify that the pMMO concentrations remained constant, and that therewas no leaching through the course of the study. As shown in FIG. 11A,the activity between assay cycles 1 to 5 remained close to the initialactivity (˜80 nmol MeOH min⁻¹mg⁻¹) and then gradually decreased to ˜45nmol MeOH min⁻¹mg⁻¹ after 20 cycles. The error bars correspond to thestandard deviation from the average of four replicates. FIG. 11B showsthe cumulative methanol produced from these 20 consecutive reactions ofPEG-pMMO compared to a single reaction of membrane bound pMMO.Immobilization of fully active pMMO in a material allowed the facileproduction of 10 fold more methanol per protein than could be producedwith membrane bound pMMO (which can only be reused with painstakingrepeated centrifugation and rinsing steps).

c. Continuous Flow-Through Bioreactor

Establishing that that the PEG-pMMO material could be reused with nomeasureable protein leaching indicated that the material would beamenable for use in a bench-scale continuous flow reactor. A designwhere the pMMO material is suspended between gas and liquid reservoirswas discovered herein as desirable given that pMMO acts upon gas phasereactants and generates liquid phase. However, PEG-pMMO, and hydrogelsin general, are mechanically brittle and difficult to handle when moldedas thin membranes. Accordingly, the PEG-pMMO material was embedded intoa three dimensional silicone lattice (printed using Direct Ink Write) inorder to greatly increase the mechanical stability and to easily tunethe size and shape of the hydrogel for use in a continuous reactor (FIG.12A). As discussed in greater detail below, the lattice was constructedof 250 micron silicone struts and contained 250 micron void spaces (50%porosity) which were then infilled with PEGDA 575, crude pMMO membranepreparations, and photoinitiator and crosslinked in place withultraviolet light. Two such lattice structures, thin and thick, weredesigned to compare effects of PEG-pMMO surface area to volume ratio onmethanol production. The surface area to volume ratio of thin vs. thickfor these experiments was 5 to 1. The silicone lattice structureincreases the bulk gas permeability of the materials, since siliconepermeability is at least 50 times greater than the PEGDA hydrogelpermeability.

The resulting hybrid silicone-PEG-pMMO lattice materials weremechanically robust, allowing the suspension of the PEG-pMMO lattice of1 millimeter thickness between gas and liquid reservoirs in aflow-through reactor. A schematic of the reactor cross section is shownin FIG. 12A. With this configuration, a methane/air gas mixture wasflowed on one side of the lattice and the NADH was introduced on theother side, while continuously removing and collecting methanol inbuffer. In order to determine the length of time the membrane could becontinuously used, the cumulative methanol produced per mg of enzyme wasmeasured at 25° C. at 30 min intervals in the thick lattice over thecourse of 5.5 hours. The methanol production rate (slope of methanol vs.time curve) was stable for about 2.5 hours, and declined gradually overthe next 3 hours. In order to evaluate whether the geometry of PEG-pMMOmaterial influenced methanol production races, reactor outlet fractionsfrom reactors containing the thin and thick lattices were compared at 15min intervals at 45° C., over the course of two hours (FIG. 12B) intriplicate. The methanol concentrations produced in the flow reactorwere on average 12 and 6% of what was predicted, for thin and thicklattices, respectively, based upon analyte flow rates and an assumedpMMO activity of 80 nmol MeOH min⁻¹mg⁻¹. The low concentration valuesrelative to predicted values may be due to lower actual pMMOconcentrations in the material than was calculated. As shown in FIG.12B, the methanol produced (per mg of protein) by the thin membrane wasdouble that produced by the thick membrane over the course of the firsthour. Over the following hour, the methanol production rate by the thinmembrane declined relative to that of the thick membrane; after twohours the average total methanol produced by the thin membrane was 1.5times higher than that produced by the thick membrane. The resultsdemonstrate that the ability to tune the geometry of immobilized pMMO,even at the millimeter scale, impacts the performance of thebiocatalytic material.

d. Direct Printing of PEG-pMMO Hydrogels

Projection microstereolithography (PμSL) allows three dimensionalprinting of light-curable materials by projecting a series of images onthe material, followed by changing the height of the stage at discreteincrements, with micron-scale resolution in all three dimensions.Therefore, it was an ideal technique for directly printing the PEG-pMMOmaterial and determining whether changing geometrical features of thematerial at these length scales can influence activity. PμSL was thusused to print PEG-pMMO lattice structures with increased surface area tovolume ratio due to 100 μm² vertical channels corresponding to ˜15% voidvolume. In this experiment, the pMMO concentration of 5 mg/ml did notattenuate the light enough for highest resolution printing; consequentlyfeature resolution was reduced in the z-direction and each layer ofprinted pMMO was exposed to multiple exposures to UV light. The pMMOactivity in the printed cubic lattices with a total volume of about 27mm³, which took approximately 50 min to print using PμSL, wasreproducible but modest at 29 nmol MeOH min⁻¹mg⁻¹. The reduction inactivity compared to crude pMMO is likely due to the duration of theprinting at room temperature as well as the overexposure of pMMO to UVduring curing. However, the cubic lattices retained about 85% of theenzyme based on the solid volume of the lattice (23 mm³) correspondingto the highest protein loading that was have achieved. While not wishingto be bound by any theory, it is thought that this high retention waslikely due to higher cross-linking efficiency.

Since the lattice geometry did not permit precise tuning of surface areato volume ratios, due to bending of lattice struts under water surfacetension, a different PμSL tool designed to generate larger parts wasused to print solid and hollow PEG-pMMO cylinders with surface area tovolume ratios ranging from 1.47-2.33 and diameters ranging from of 1-5mm. The hollow tube geometry may allow more facile diffusion ofreactants because both the inner and outer surfaces of the cylindricalmaterials would be exposed. The total print time for an array ofcylinders using the large-area PμSL tool was significantly reduced to ˜1min by eliminating z-axis resolution, and the pMMO concentration wasreduced to 2.3 mg/ml to allow UV light penetration through the 1.5-3 mmdepth of the resin. Remarkably, the activity of pMMO in the hydrogelsincreased with greater surface area to volume ratios as shown in FIG. 13, with the highest ratio of 2.33 resulting in an average activity of128+/−14 nmol MeOH min⁻¹mg⁻¹ per cylinder, which corresponds to thehighest reported physiological activity of membrane bound pMMO. Thecylinders of the lowest ratio, 1.47, had an average pMMO activity of67+/−3 nmol MeOH min⁻¹mg⁻¹. It should also be noted that the cylinderswith the lowest surface area to volume ratio were only 1.5 mm in heightand therefore completely submerged in the liquid phase during theactivity assay, whereas all other cylinders tested were 3 mm in heightand only partially submerged during the assay. Hydrogels protruding fromthe liquid allowed a direct interface between the gas phase andPEG-pMMO. This exposed interface likely increased the methaneconcentration in the PEG-pMMO material since the solubility of methanein PEG is several times higher than that in water. On average, 38% ofthe protein was encapsulated, although it was variable depending on thedimensions of each cylinder (27-54%). These results, combined with theresults from the continuous flow reactor, indicate that an optimal pMMOmaterial design may be hierarchical,with the smallest feature sizes atthe micron scale.

Specific Methods

a. Materials

Reagents for buffers (PIPES, NaCl, and NaOH), HPLC grade methanol(≥99.9% purity), polyethylene glycol diacrylate 575 (PEGDA 575), and thecross-linking initiator, 2-hydroxy-2-methylpropiophenone (Irgacure®1173), was purchased from Sigma-Aldrich (St. Louis, Mo.). All reagentswere used as received. Methane gas (99.9% purity) was obtained fromMatheson Tri-gas, Inc. (Basking Ridge, N.J.). pMMO concentrations weremeasured using the DC™ protein assay purchased from Bio-Rad (Hercules,Calif.). Lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP)photoinitiator was synthesized following a procedure known in the art.

b. pMMO: Cell Growth and Membrane Isolation

Methylococcus capsulatus (Bath) cells were grown in 12-15 Lfermentations. M. capsulatus (Bath) cells were grown in nitrate mineralsalts medium (0.2% w/v KNO₃, 0.1% w/v MgSO₄.7H₂O and 0.001% w/vCaCl₂.2H₂O) and 3.9 mM phosphate buffer, pH 6.8, supplemented with 50 μMCuSO₄.5H₂O, 80 μM NaFe(III) EDTA, 1 μM Na₂MoO₄.2H₂O and trace metalssolution. Cells were cultured with a 4:1 air/methane ratio at 45° C. and300 rpm. Cells were harvested when the A₆₀₀ reached 5.0-8.0 bycentrifugation at 5000×g for 10 min. Cells were then washed once with 25mM PIPES, pH 6.8 before freezing in liquid nitrogen and storing at −80°C. Frozen cell pellets were thawed in 25 mM PIPES, pH 7.2, 250 mM NaClbuffer (herein referred to as pMMO buffer) and lysed by microfluidizerat a constant pressure of 180 psi. Cell debris was then removed bycentrifugation at 20,000-24,000×g for one hr. The membrane fraction waspelleted by centrifugation at 125,000×g for one hour and washed 3 timeswith pMMO buffer before freezing in liquid nitrogen and storing at −80°C. Final protein concentrations were measured using the Bio-Rad DC™assay. Typical storage concentrations ranged from 20-35 mg/ml.

c. Formation of the PEG-pMMO Hydrogels

Prior to preparation of the PEG-pMMO hydrogels, frozen as-isolated crudemembranes from M. Capsulatus (Bath) (herein referred to asmembrane-bound pMMO) was thawed at room temperature and used within 5hours of thawing. Thawed membrane-bound pMMO (50-500 μg) was then mixedwith PEGDA 575 in pMMO buffer at room temperature to form liquid PEG andpMMO suspensions having a final volume of 50 μl and 10-80 (v/v %) PEGDA575. A photoinitiator (not shown in FIG. 9 ) was included in thesuspension at 0.5 vol % with respect to PEGDA 575. The suspension wasmixed by pipetting to homogeneity and then transferred to a 1 ml syringewith the tip removed. The syringe was then immediately placed under UVlight at 365 nm, 2.5 mW/cm² intensity, for 3 min. After the UV exposure,the 50 μl PEG-pMMO hydrogel block was slowly pushed out of the syringeonto a kimwipe where it was gently blotted and then rinsed twice in pMMObuffer to remove unreacted reagents.

d. Activity Assay

All reactions were carried out in 2 ml glass reaction vials in pMMObuffer with 6 mM NADH as a reducing agent. Vials with 50-500 μg pMMO in125 μl buffer solution were used as controls. For the immobilized enzymesamples, each 50 μl PEG-pMMO hydrogel block was placed in a vial andpartially submerged in 75 μl buffer solution immediately after curingand rinsing. 1 ml of headspace gas was removed from each vial using a 2ml gas tight glass syringe and replaced with 1 ml of methane, then thereaction vial was immediately placed in a heating block set at 45° C.and incubated for 4 min at 200 rpm. After 4 min, the samples were heatinactivated at 80° C. for 10 min. Samples were then cooled on ice for 20min and pMMO control vials were centrifuged to remove the insolublemembrane fraction. For the cyclic activity assays using the PEG-pMMOimmobilized enzyme, the reaction was stopped by opening and degassingthe head space and immediately removing the solution for GC analysis.The block was then rinsed three times with 1 ml of pMMO buffer per washand the assay was repeated. The amount of methanol generated during thereaction was measured by gas chromatography/mass spectrometry (GC/MS)analysis using an Agilent Pora-PLOT Q column and calibration curves weregenerated from methanol standards.

e. pMMO Flow Reactor

A simple cubic polydimethyl siloxane (PDMS) lattice with 250 micronstruts and 250 micron spacing was printed using Direct Ink Write asdescribed to provide methane permeability throughout the PEG materialand to provide mechanical support. A top layer of 50 micron thick PDMSwas fabricated by spin-coating Dow Corning SE-1700 PDMS diluted intoluene on a hydrophobized silicon wafer. This thin PDMS membraneprevented leakage of liquid through the membrane but provided gaspermeability. Two different flow cell geometries were fabricated usingpolycarbonate plastic: a flow cell for a higher surface area, thinlattice (1.25 cm wide by 3 cm long) and a lower surface area, thicklattice, 1.25 by 1.25 cm. The thin lattice was 6 layers thick, and thethick lattice had 16 layers. The lattices were made hydrophilic bytreating them in air plasma for 5 minutes followed by storage indeionized water. To incorporate the pMMO into the lattices, a 10 vol %concentration of PEGDA 575 was mixed with crude pMMO membranepreparations to a final concentration of 5 mg/ml pMMO. Two hundredmicroliters of the pMMO/PEGDA mixture were pipetted into the lattice andcured with 365 nm UV light at 2.5 mW/cm² intensity for 4 min, formingthe mixed polymer (PEG/PDMS) membrane. The final concentration of pMMOin the lattices was calculated, rather than directly quantified using aprotein assay, due to difficulties in quantifying the material in thelattice. The membrane was then loaded into the cell and rinsed withbuffer to remove any unpolymerized material. The flow cell was placed ona hot plate calibrated with thermocouple so that the membrane wouldreach either 25 or 45 degrees ° C. An NADH/buffer solution (4 mg/ml NADHin PIPES pH 7.2) was prepared as the liquid phase in a 5 ml syringe, andthe gas phase was prepared as 50% methane and 50% air loaded into agas-tight 50 ml syringe. The syringes were loaded into Harvard Apparatussyringe pumps and the gas and liquid were delivered at 0.5 and 0.75 mlper hour, respectively. The gas outlet tubing was kept under 2 cm waterpressure during the reaction. Fractions of liquid were collected intoGC/MS autosampler vials that were kept on ice to reduce methanolevaporation and were analyzed against MeOH standards using GC/MS asdescribed above. Methanol contamination was present in the NADH/buffersolutions, and this concentration was subtracted from the total detectedin each fraction by GC/MS. No methanol contamination was found in thewater used to store the PDMS. The data shown in FIG. 12B representcumulative methanol (where the quantity of methanol produced in eachfraction was added to the previous samples). Each experiment was done intriplicate; the error bars represent a standard deviation.

f. 3D Printing of PEG-pMMO Hydrogels

The printing resin was prepared with 20 vol % PEGDA 575, 10 mg/ml LAPinitiator, and 2.3-5 mg/ml crude pMMO in buffer. Using projectionmicrostereolithography (PμSL), hydrogel blocks were printed in a cubiclattice with 100 um open channels spaced 100 um apart and sizedimensions from 1-3 mm. Solid and hollow cylinders of the same resinformulation were printed using the large area PμSL (LA PμSL) system. Thecylinders had an inner diameter of 1-2.5 mm, an outer diameter of 3-5mm, and were 1.5-3 mm high. The resin was cured with a 395 nm diode withboth PμSL and LA PμSL but the intensity and exposure time varied betweenthe systems, ranging from 11.3-20 W/cm² and 15-30 seconds per layer,respectively. Resin and printed hydrogels were stored on ice before andafter the printing process. The pMMO activity assay was carried out asdescribed above at 45° C. for 4 minutes. The methanol concentration ofthe activity assay and protein content of the printed hydrogels weremeasured as described above.

Microcapsule Embodiments

The foregoing descriptions primarily reference block polymer and/orcopolymer networks as the structural arrangement most suitable forcarrying out the respective chemical reactions. Those having ordinaryskill in the art will appreciate that in particular applications the useof a biocatalytic microcapsule provides additional advantages and/orfunctionalities beyond those described above regarding polymer/copolymernetworks.

However, it should be noted that the following descriptions ofmicrocapsule embodiments may employ any of the foregoing compositions,structures, techniques, etc. described with reference to block polymerand/or copolymer networks. In some approaches for example the polymericshell of the microcapsules may be or comprise a block polymer/copolymernetwork, which may comprise any combination of suitable polymers asdescribed above with reference to block polymer/copolymer embodiments.In other approaches, the microcapsules may be embedded in a polymericnetwork as described hereinabove.

Utilizing biocatalytic microcapsules as described in further detailbelow enables production of highly efficient gas conversion systems. Thepresently disclosed biocatalytic microcapsules may be configured inpacked stationary or moving beds, as well as fluidic beds, or embeddedin meshes or adhesive compositions, allowing a broad applicability tomany industrial uses requiring gas conversion (such as greenhouse gasemission reductions, food preservation, etc.).

More specifically, by encapsulating all necessary reagents/components tocarry out gas conversion in a robust polymeric shell permeable to thegas species targeted for conversion, available surface area foradsorbing the target gases may be dramatically increased (e.g. on theorder of approximately ten-fold relative to conventional stir tankapproaches). Improvements to mass transport across the polymeric shellrelative to solvation of the gases in the conventional liquid mediumfurther improve the efficiency of gas conversion. In addition, since thebiocatalyst is isolated in the interior of the polymeric shell, andincludes all necessary components/reagents for renewable catalysis, thebiocatalytic microcapsules of the presently disclosed inventive conceptsprovide improved capacity for reuse and longevity of the catalyst in itsintended application.

Further still, and also an advantageous feature of using catalystsisolated in polymeric shells, the presently described inventivemicrocapsules may be employed in dry applications, and are thereforesuitable for use as preservatives of materials sensitive to particulargases (e.g. food items are generally sensitive to ethylene and decomposemore rapidly in the presence of ethylene, by converting ethylene to e.g.ethylene oxide the ethylene-driven acceleration of the decompositionprocess may be retarded or terminated). Accordingly, biocatalyticmicrocapsules as described herein allow the use of efficient gasconversion catalysts in passive environments and applications and do notrequire a liquid medium to facilitate mass transport of the target gasesfor conversion.

Further still, by selecting appropriate polymer(s) for use in thepolymeric shell, preferred embodiments of biocatalytic microcapsulesintended for use in passive applications such as food/medicinepreservation may be non-toxic (i.e. at least to human biology).Moreover, the presently disclosed inventive microcapsules may befabricated in a much more cost-efficient manner than traditionalmaterials employed for such passive applications (e.g. palladium,platinum, and other precious metal catalysts, which typically are bothexpensive and toxic).

Accordingly, the presently disclosed inventive biocatalyticmicrocapsules represent a significant improvement to the function ofconventional gas conversion technology, as well as an extension of gasconversion to applications and environments that are not possible usingconventional techniques and technology.

These improvements are conveyed by two important, indeed criticalcomponents of the presently described inventive concepts, and include 1)a biocatalytic material, e.g. dried and reconstituted cells of suitableorganisms such as methanotrophs (or components thereof such as describedin greater detail below), which readily oxidize C1-C3 gases such asmethane, carbon monoxide, carbon dioxide, ethane, ethylene; propane;and/or propylene; combined with 2) the encapsulation of this material ina polymer shell which readily allows penetration of the gas phasereactants, and release of converted products. The size scale of thesecapsules may have a diameter in a range from approximately 10 toapproximately 1000 microns, with the polymeric shell having a thicknessin a range from approximately 5 microns to approximately 100 microns, invarious embodiments and depending on the application to which themicrocapsules are to be employed. Preferably, the capsules are sphericaland the polymer shell is either hydrophobic and gas permeable, forexample silicone, or amphiphilic, allowing transport of gasses andcharged species, e.g. following the configuration of a block copolymersuch as described hereinabove.

The biocatalytic capsules can be used in a packed bed, moving bed, orfluidized bed configuration. Alternatively, they can be immobilizedembedded in a second material, like a mesh, provided in a packetencapsulating the microcapsules but permitting gases to permeate thepacket (e.g. such as packets included in certain food packaging toprevent oxidation of food products contained therein) or added to aliquid adhesive, to create a conformal coating, e.g. on a surface or asan insert in a produce box to remove ethylene. In accordance with oneembodiment, the inventors have demonstrated the synthesis of stable,catalytically active microcapsules in the laboratory that oxidizepropylene under ambient conditions.

Several details will now be presented regarding the structure andcomposition of the inventive biocatalytic microcapsules with referenceto FIGS. 14A-14B, according to a preferred embodiment.

As shown in FIG. 14A, a microcapsule 1400 includes a polymeric shell1402 defining an internal region 1404. The polymeric shell has athickness t in a range from approximately 5 microns to approximately 100microns, and an outer diameter d in a range from approximately 10microns to approximately 1000 microns. As noted above the particularthickness and diameter of the microcapsule 1400 may be chosen based onthe particular application to which the microcapsules will be employed.

For instance, smaller microcapsules with thinner walls may be desired incertain applications where maximum surface area and mass transport aredesired, but mechanical strength is less important (e.g. in passiveapplications where the microcapsules are not subject to agitation orother mechanical stress, including packed, dry beds). In otherembodiments, e.g. food preservation, where the microcapsules may besubject to mechanical stress associated with handling of the food itemsand/or packaging the thickness of the walls may be on the higher end ofthe spectrum noted above. Skilled artisans will appreciate, upon readingthe instant descriptions, the particular thickness and/or microcapsulediameter ranges appropriate for various applications.

Moreover, the polymeric shell 1402 is preferably permeable to the targetgas(es) to be catalyzed/converted by the biocatalytic componentsincluded in the microcapsule 1400. In various embodiments, polymericshell 1402 is insoluble in aqueous solutions, and is preferably eitherhydrophobic and permeable to said target gases, or amphiphilic andconducts charged species and target gases across the polymeric shell1402, which acts effectively as a selective membrane to facilitate masstransport of the target gases from the environment to the interiorregion 1404 of the microcapsule 1400.

Even more preferably, the polymeric shell 1402 is also permeable and/orconducts the products of catalyzing/converting the target gases.Optionally, the permeability/conductivity of the polymeric shell 1402 tothe products of the catalysis/conversion reaction may be tuned byadjusting a (preferably reversible) parameter such as environmentaltemperature, pH, etc. to provide selective ability to release thereaction products under desired conditions.

For instance, target gases may be captured and converted at atmosphericconditions, but to avoid contaminating the environment withcatalysis/conversion products the polymeric shell 1402 may besubstantially (e.g. 90% or greater) impermeable to the products underatmospheric conditions. However, by elevating the temperature of themicrocapsules 1400, the products may be released. Such a scheme allowsisolation of the microcapsules having the catalysis/conversion productsdisposed therein (e.g. in a containment or collection system/facility)and selective release of such products so as to avoid simplyreintroducing e.g. a reversible product which may be converted back intothe undesired target gas in the operational environment.

In various approaches, the polymeric shell 1402 may include crosslinkedpolymers formed from one or more polymer precursors. Crosslinking andformation of the polymeric shell will be discussed in further detailbelow regarding FIG. 15 . In various embodiments, the polymer precursorsmay include any one or more of the following: polydimethylsiloxane(PDMS), polyethylene glycol (PEG); polyethylene glycol diacrylate(PEGDA); hexanediol diacrylate (HDDA), polyvinyl alcohol (PVA);poly(lactic acid) (PLA); polyimide; poly(2-methyl-2-oxazoline) (PMOXA);poly(ether ether ketone) (PEEK); cellulose acetate; polypropylene (PP);3-[tris(trimethylsiloxy)silyl]propyl methacrylate; trimethylolpropanetrimethacrylate; 2-Hydroxy-2-methylpropiophenone; and silicone acrylate.

In one preferred approach, the polymeric precursors include a mixture ofthe 3-[tris(trimethylsiloxy)silyl]propyl methacrylate; thetrimethylolpropane trimethacrylate; and the2-Hydroxy-2-methylpropiophenone. More preferably, the mixture includes3-[tris(trimethylsiloxy)silyl]propyl methacrylate present in an amountfrom about 70 wt % to about 90 wt %; trimethylolpropane trimethacrylatepresent in an amount from about 10 wt % to about 30 wt %; and2-Hydroxy-2-methylpropiophenone present in an amount from about 0.1 wt %to about 10 wt %.

In a particularly preferred embodiment, the polymer precursor mixturecomprises 79.5 wt % 3-[tris(trimethylsiloxy)silyl]propyl. methacrylate(preferably containing mono methyl ether hydroquinone (MEHQ) in anamount ranging from about 200-800 PPM as stabilizer, 98% pure orgreater), 19.5 wt % trimethylolpropane trimethacrylate (preferablycontaining 250 ppm monomethyl ether hydroquinone as an inhibitor,technical grade); and 1 wt % 2-hydroxy-2-methylpropiophenone (97% pureor greater). Utilizing this mixture the inventors were able to reliablyfabricate biocatalytic microcapsules 1400 having a diameter ofapproximately 300 microns, as shown in FIG. 14B according to oneembodiment.

With continuing reference to FIG. 14A, microcapsule 1400 also includesone or more biocatalysts contained in the interior region 1404 of thepolymeric shell 1402. Although the embodiment of FIG. 14A depicts aplurality of distinct types of biocatalysts, including enzymes 1408,enzyme cofactors 1410 (which may or may not be associated with enzymes);cell membrane fragments 1412, reconstituted whole cells 1414, andcytosolic components 1416, it should be understood that in variousapproaches microcapsules 1400 may include any one of the foregoing, orany combination thereof without departing from the scope of thepresently disclosed inventive concepts. Example biocatalysts include:membrane fractions, cytosolic components, whole dried/reconstituted,and/or live cells such as soil microbes Methanococcus Capsulatus, andRalstonia Eutropha, or the acetogenic anaerobes such as Acetobacteriumwoodii. Cofactors may include any combination of formate, NADH,duroquinol, etc. as would be appreciated by a person having ordinaryskill in the art upon reading the present disclosures. Additionally, insome embodiments reducing agents can be supplied inside the capsule, thereducing agents being derived from proteins such as hydrogenase (whichadvantageously regenerate reducing equivalents with the addition ofhydrogen) or methanol dehydrogenase (which regenerate reducingequivalents from the conversion of methanol product to formaldehyde)and/or formate dehydrogenase.

Preferably, the biocatalytic components included in the interior region1404 of microcapsule 1400 include at least an entire proteome of one ormore organism(s) adapted to catalyze or convert target gases for theapplication to which the microcapsules 1400 are to be employed, or atleast those portions of a proteome of the organism that include proteinsinvolved in the catalysis or conversion of such target gases.

The biocatalytic components 1408-1416 may be suspended in an aqueousbuffer solution 1406 also disposed in the interior region of themicrocapsule. Preferably, the buffer includes a reducing agent, but asnoted above one advantage of using reconstituted whole cells is the lackof a need to include additional or separate reducing agent. Thereconstituted whole cells provide the requisite reducing agent, if any,improving the longevity of the biocatalyst included in the microcapsule1400. In one embodiment, the reducing agent comprises formate, and maybe included in the buffer in an amount ranging from about 1 to about 100millimolar.

Regardless of whether the buffer includes additional reducing agent, thebuffer solution is preferably aqueous and has a pH in a range fromapproximately 4.0 to approximately 10.0.

The use of reconstituted whole cells (or, in various embodiments selectsubsets of cell components, such as an entire proteome of a particularorganism; select enzymes and/or associated cofactors, cell membranefragments and associated proteins; cytosolic cell components such ascytosolic proteins, organelles, etc.) encapsulated in a polymeric shellconveys several advantages in the context of the presently describedinventive microcapsules.

First, such microcapsules include all necessary reagents and/orcomponents to carry out the conversion reaction, and may not require theaddition of any reducing agent to the microcapsule (typically, areducing agent is necessary to provide renewable catalytic activity)since the reconstituted organism may advantageously include anappropriate reducing agent naturally.

Second, encapsulating such biocatalysts in a polymeric shell, e.g. asopposed to a lipid bilayer encapsulating a liposome or enzyme mixtureper to conventional approaches, provides drastically improved stabilityof the biocatalyst and broader applicability to the biocatalyst (e.g.beyond stir-tank embodiments and including dry and/or passiveapplications, as well as high throughput configurations such asfluidized beds, in which lipid bilayer-based configurations wouldcollapse).

To leverage the most efficient catalytic pathways for gas conversion,embodiments of the presently disclosed inventive concepts preferablyutilize reconstituted whole cells of an organism adapted to carrying outthe conversion reaction. In the context of C1-C3 gases, e.g. methane,ethane, ethylene, propylene, etc. as discussed herein, methanotrophicorganisms are a generally suitable class of organism for conducting gasconversion.

The cells may be lyopholized and reconstituted in an appropriate(preferably aqueous) buffer to form a suspension of the biocatalyst(s).This suspension may be emulsified in a mixture of polymer precursors,which are in turn emulsified in an appropriate aqueous carrier fluid,and the polymer precursors may be cured to form the polymeric shellhaving the biocatalyst suspension disposed therein. Formation ofmicrocapsules will be described in further detail below with referenceto FIGS. 15-16 .

Turning now to FIG. 15 , a simplified top-down schematic of an exemplaryapparatus 1500 for producing biocatalytic microcapsules is shown,according to one embodiment. The apparatus 1500 includes a bath 1502filled with an aqueous carrier fluid 1504 into which droplets 1508 of amicrocapsule precursor material are formed/extruded/deposited using anappropriate droplet generator 1506 of any type known in the art thatwould be appreciated by a skilled artisan as suitable for generatingdroplets 1508 having characteristics as described herein. In variousembodiments, such characteristics of droplets 1508 may include, but arenot limited to: emulsification structure (including but not limited to adouble emulsion structure between aqueous carrier fluid 1504, polymericprecursors, and biocatalytic components in the droplets 1508, andthickness of the polymer precursor phase), composition of polymerprecursors and biocatalytic components, and droplet size (e.g.diameter).

In preferred approaches, each of the droplets 1508 aredeposited/extruded, etc. into the aqueous carrier fluid 1504 in the formof an emulsion, more specifically a suspension of one or morebiocatalytic components (e.g. 1408-1416 as shown in FIG. 14A), thebiocatalytic component suspension being emulsified in a polymerprecursor layer comprising one or more polymer precursors.

As noted above, and in various embodiments, the polymer precursors mayinclude any one or more of the following: polydimethylsiloxane (PDMS);polyethylene glycol (PEG); polyethylene glycol diacrylate (PEGDA);hexanediol diacrylate (HDDA); polyvinyl alcohol (PVA); poly(lactic acid)(PLA); polyimide; poly(2-methyl-2-oxazoline) (PXMOA); poly(ether etherketone) (PEEK); cellulose acetate; polypropylene (PP);3-[tris(trimethylsiloxy)silyl]propyl methacrylate; trimethylolpropanetrimethacrylate; 2-Hydroxy-2-methylpropiophenone; and silicone acrylate.

The polymer precursors may optionally include stabilizers such as monomethyl ether hydroquinone (MEHQ), etc. to facilitate proper fluiddynamics during deposition/extrusion of droplets 1508 including but notlimited to viscosity, shear, flow rate, surface tension, etc. and thuspreserve the emulsion between the polymer precursor layer and thebiocatalytic components.

The polymer precursors may additionally and/or alternatively compriseone or more inhibitors, e.g. photoinhibitors configured to preventspontaneous crosslinking of polymer precursors in response to exposureto ambient light. The inhibitors are preferably selected based on thecuring process and conditions to be employed to convert the polymerprecursors to a polymeric shell.

In one embodiment, polymer precursor fluid includes: approximately 79.5wt % 3-[Tris(trimethylsiloxy)silyl]propyl methacrylate (optionally butpreferably including MEHQ as stabilizer, 98% purity), approximately 19.5wt % Trimethylolpropane trimethacrylate (optionally but preferablyincluding 250 ppm monomethyl ether hydroquinone as inhibitor, technicalgrade), 1 wt % 2-Hydroxy-2-methylpropiophenone (97% purity). Anotheroption is a commercially available silicone acrylate (but used forcompletely different purposes), e.g. TEGO RAD 2650.

In one preferred approach, the polymeric precursors include a mixture ofthe 3-[tris(trimethylsiloxy)silyl]propyl methacrylate; thetrimethylolpropane trimethacrylate; and the2-Hydroxy-2-methylpropiophenone. More preferably, the mixture includes3-[tris(trimethylsiloxy)silyl]propyl methacrylate present in an amountfrom about 70 wt % to about 90 wt %; trimethylolpropane trimethacrylatepresent in an amount from about 10 wt % to about 30 wt %; and2-Hydroxy-2-methylpropiophenone present in an amount from about 0.1 wt %to about 10 wt %.

In a particularly preferred embodiment, the polymer precursor mixturecomprises 79.5 wt % 3-[tris(trimethylsiloxy)silyl]propyl methacrylate(preferably containing mono methyl ether hydroquinone (MEHQ) in anamount ranging from about 200-800 PPM as stabilizer, 98% pure orgreater), 19.5 wt % trimethylolpropane trimethacrylate (preferablycontaining 250 ppm monomethyl ether hydroquinone as an inhibitor,technical grade); and 1 wt % 2-hydroxy-2-methylpropiophenone (97% pureor greater).

Polymer precursors of the variety mentioned above may be obtainedcommercially and prepared (e.g. by solvating appropriate particles in anappropriate solvent) using techniques known in the art. The polymerprecursor mixture may also be combined with the biocatalyst suspension,and an emulsion thereof generated, using techniques known in the art.This emulsion may be provided to the droplet generator 1506 fordelivering droplets 1508 to the aqueous precursor fluid.

Upon delivery to the aqueous carrier fluid 1504, the droplets form adouble emulsion in which the biocatalyst suspension is emulsified in thepolymer precursor fluid, and the polymer precursor fluid is emulsifiedin the aqueous carrier fluid 1504. To facilitate forming the doubleemulsion, in preferred approaches the aqueous carrier fluid 1504preferably includes water present in an amount from about 50 wt % toabout 60 wt %; glycerol present in an amount from about 30 wt % to about40 wt %; and polyvinyl alcohol present in an amount from about 1 wt % toabout 5 wt %. More preferably, according to one embodiment the aqueouscarrier fluid 1504 comprises approximately 58 wt % water, approximately40 wt % glycerol, and approximately 2 wt % polyvinyl alcohol. Mostpreferably, monomers of the PVA are independently characterized by amolecular weight in a range from approximately 13,000-23,000 g/mol, andare approximately 87-89% hydrolyzed.

The droplets 1508 travel through the bath 1502 along a prevailing flowdirection and are carried toward a curing region 1512 in which thepolymer precursor layer of the droplets 1508 are cured to form apolymeric shell such as polymeric shell 1402 as shown in FIG. 14A. Inpreferred approaches, and in accordance with the embodiment of FIG. 15 ,the curing process involves photocuring the polymer precursors, e.g. viaUV crosslinking. Accordingly, apparatus 1500 may include a light source1510 configured to emit light having predetermined characteristics (e.g.a wavelength suitable to initiate crosslinking in the polymer precursorsof the droplets 1508) within the curing region 1512.

Droplets flowing through the curing region 1512 are exposed to lightfrom the light source 1510, causing the polymer precursors to crosslinkand solidify, resulting in biocatalytic microcapsules 1400. Themicrocapsules 1400 may continue to flow along the flow direction of thebath 1502 toward an outlet of the apparatus 1500 (not shown).

In some embodiments, microcapsules 1400 may be stored in an appropriatebuffer, preferably an aqueous buffer excluding target gases forsubsequent catalysis/conversion. Optionally, the microcapsules may beremoved from the aqueous carrier fluid 1504 and washed (e.g. in water)and/or dried for subsequent use in an embedded matrix, mesh, adhesive,etc. for dry applications such as food preservation.

Accordingly, and with reference to FIG. 16 , a preferred embodiment of amethod 1600 of forming microcapsules for selective catalysis of gases isshown. The method 1600 may be performed in any suitable environment,including but not limited to the apparatus as shown in FIG. 15 , withoutdeparting from the scope of the present disclosures. Moreover, themethod 1600 may include any number of additional or alternativeoperations than shown in FIG. 16 , in alternate embodiments, so long asthe result is formation of biocatalytic microcapsules as describedherein.

The present method 1600 may be implemented in conjunction with featuresfrom any other embodiment listed herein, such as those described withreference to the other FIGS. Of course, the method 1600 and otherspresented herein may be used in various applications and/or inpermutations, which may or may not be specifically described in theillustrative embodiments listed herein.

Returning now to FIG. 16 , in accordance therewith method 1600 includesoperation 1602, in which at least one biocatalyst is emulsified in apolymer precursor mixture. The emulsion may be prepared using anysuitable technique that would be appreciated as suitable by a skilledartisan after reading the instant descriptions. Preferably, thebiocatalyst(s) is/are suspended in an appropriate buffer fluid, whichmay or may not include reducing agent, and is preferably an aqueousbuffer. The biocatalyst suspension may include any number or type ofbiocatalytic components as described herein, e.g. with reference to FIG.14A above. The polymer precursor mixture may have any composition asdescribed hereinabove, or equivalents thereof that would be appreciatedby skilled artisans upon reading the present disclosure.

In operation 1604, method 1600 involves emulsifying the polymerprecursor mixture in an aqueous carrier solution. The process ofemulsifying the polymer precursor mixture, which is preferably presentin the form of a droplet such as droplets 1508 as shown in FIG. 15 ,involves controlling fluid dynamics within a suitable bath containingthe aqueous carrier solution into which the droplets (having the polymerprecursor mixture disposed in an outer layer thereof) areformed/deposited. In addition, the composition of the aqueous carriersolution and the polymer precursor mixture should be formulated so thatan emulsion therebetween may be formed. Accordingly, polymer precursormixture and aqueous carrier solution may have a composition as describedelsewhere herein, according to various embodiments.

With continuing reference to FIG. 16 , in operation 1606 of method 1600polymer precursor(s) of the polymer precursor mixture are cured, e.g.via crosslinking, to form a polymeric shell therefrom. This results in aplurality of microcapsules each independently comprising: a polymericshell permeable to one or more target gases; and at least onebiocatalyst disposed in an interior of the polymeric shell, e.g. inaccordance with microcapsules 1400 as shown in FIGS. 14A-14B.Preferably, the curing involves projection microstereolithography.

Applications/Uses

Embodiments of the present invention may be used in a wide variety ofapplications, and potentially any industrial application requiring moreefficient and higher-throughput use of enzymes to catalyze chemicalreactions. Illustrative applications in which embodiments of the presentinvention may be used include, but are not limited to, fuel conversion(e.g., natural gas to liquid fuel), chemical production, pharmaceuticalproduction, and other processes where a chemical conversion is catalyzedby enzymes, especially at phase boundaries (e.g., reaction involving agas and a liquid, polar and non-polar species, aqueous and non-aqueousspecies, etc.).

In accordance with several embodiments, and with reference to method1700 as shown in FIG. 17 , the presently disclosed inventivebiocatalytic microcapsules may be utilized in techniques for catalyzingone or more target gases. The method 1700 may be performed in anysuitable environment, including but not limited to stir tanks, fluidizedbeds, packed beds, moving beds, embedded meshes and/or adhesives, etc.,without departing from the scope of the present disclosures. Moreover,the method 1700 may include any number of additional or alternativeoperations than shown in FIG. 17 , in alternate embodiments, so long asthe result is catalysis/conversion of target gases (e.g. C1-C3 gases)using biocatalytic microcapsules as described herein.

In one embodiment, method 1700 includes exposing a plurality of thebiocatalytic microcapsules to the one or more target gases in operation702. Importantly, the biocatalytic microcapsules each independentlycomprise: a polymeric shell permeable to the one or more target gases;and at least one biocatalyst disposed in an interior of the polymericshell.

The exposure may be performed in any suitable manner, such as passivelyallowing target gases to flow over or through the microcapsules (whichmay optionally be arranged in a bed, mesh, adhesive, etc. activelypassing target gases through the microcapsules (e.g. driven by pressure,via bubbling gases through a fluidized bed, agitation of themicrocapsules, movement of microcapsules within a bed or use of a movingbed, etc.) depending on the application in question.

For example, for a carbon dioxide capture application packed beds andfluidized beds are most viable. For smaller scale industrialapplications such as ethylene or methane conversion fixed beds, orpacked beds are preferred. Generally, avoiding moving parts for smallerapplications is desirable to minimize maintenance and energyconsumption, while for larger scale applications a fluidized bed is apreferred, energy efficient configuration. For applications such as foodand medicine preservation, involving e.g. capture of ethylene, dry,small scale configurations such as microcapsules embedded in meshes oradhesives are desired to facilitate use of the microcapsules in thepackaging containing the food or medicine to be preserved. Of course, invarious applications different configurations of the microcapsules maybe employed without departing from the scope of the present disclosures.

The target gases are preferably C1-C3 compounds, e.g. methane, carbonmonoxide, carbon dioxide, ethane, ethylene; propane; and/or propylene,in various embodiments, and are preferably catalyzed by one or morebiocatalytic components selected from: one or more enzymes configured tocatalyze the one or more target gases; one or more enzyme cofactors; oneor more cell membrane fragments; one or more cytosolic cell components;and reconstituted whole cells.

Various embodiments herein have been described with reference tocatalysis or conversion of target gases using biocatalysts. It should beunderstood that catalysis, conversion, etc. of target gases may includeany suitable chemical modification such as reduction or oxidation of thetarget gases into another species (gas or liquid, preferably). In moreembodiments, catalysis/conversion may include capturing such targetgases, which may be reversibly released into a collection environment,e.g. in the case of carbon dioxide. Skilled artisans reading the presentdisclosures will appreciate other equivalent suitable forms of catalysisand/or conversion of target gases to which the presently describedinventive microcapsules may be applied.

It should be noted that methodology presented herein for at least someof the various embodiments may be implemented, in whole or in part, incomputer hardware, software, by hand, using specialty equipment, etc.and combinations thereof.

Moreover, any of the structures and/or steps may be implemented usingknown materials and/or techniques, as would become apparent to oneskilled in the art upon reading the present specification.

The inventive concepts disclosed herein have been presented by way ofexample to illustrate the myriad features thereof in a plurality ofillustrative scenarios, embodiments, and/or implementations. It shouldbe appreciated that the concepts generally disclosed are to beconsidered as modular, and may be implemented in any combination,permutation, or synthesis thereof. In addition, any modification,alteration, or equivalent of the presently disclosed features,functions, and concepts that would be appreciated by a person havingordinary skill in the art upon reading the instant descriptions shouldalso be considered within the scope of this disclosure.

While various embodiments have been described above, it should beunderstood that they have been presented by way of example only, and notlimitation. Thus, the breadth and scope of an embodiment of the presentinvention should not be limited by any of the above-described exemplaryembodiments, but should be defined only in accordance with the followingMaims and their equivalents.

What is claimed is:
 1. A microcapsule for selective catalysis of gases,the microcapsule comprising: a polymeric shell permeable to one or moretarget gases; and at least one biocatalyst disposed in an interior ofthe polymeric shell, the at least one biocatalyst being operable toconvert a C1-C3 hydrogen-comprising target gas to a product, wherein theC1-C3 hydrogen-comprising target gas is selected from the groupconsisting of: methane, ethane, ethylene, propane, and propylene.
 2. Themicrocapsule as recited in claim 1, wherein the at least one biocatalystcomprises one or more enzymes configured to catalyze the C1-C3hydrogen-comprising target gas.
 3. The microcapsule as recited in claim1, wherein the at least one biocatalyst includes reconstituted wholecells.
 4. The microcapsule as recited in claim 1, the at least onebiocatalyst comprising one or more biocatalytic components selected fromthe group consisting of: one or more cell membrane fragments; and one ormore cytosolic cell components.
 5. The microcapsule as recited in claim1, wherein the at least one biocatalyst includes live cells.
 6. Themicrocapsule as recited in claim 1, wherein the at least one biocatalystis dispersed throughout the interior of the polymeric shell.
 7. Themicrocapsule as recited in claim 1, wherein the polymeric shell ispermeable to products of catalyzing and/or converting the C1-C3hydrogen-comprising target gas.
 8. The microcapsule as recited in claim1, comprising a buffer disposed in the interior of the polymeric shell,wherein the at least one biocatalyst is suspended in the buffer.
 9. Themicrocapsule as recited in claim 1, wherein the at least one biocatalystselectively oxidizes the C1-C3 hydrogen-comprising target gas to formthe product.
 10. A product comprising a plurality of microcapsules forselective catalysis of gases, each microcapsule comprising: a polymericshell permeable to one or more target gases; at least one biocatalystdisposed in an interior of the polymeric shell, the at least onebiocatalyst comprising one or more biocatalytic components selected fromthe group consisting of: live cells, one or more cell membranefragments, one or more cytosolic cell components, and reconstitutedwhole cells; and a buffer disposed in the interior of the polymericshell, wherein the at least one biocatalyst is suspended in the buffer.11. The product as recited in claim 10, wherein the buffer comprises areducing agent.
 12. The product as recited in claim 2, wherein the atleast one biocatalyst comprises one or more enzymes configured tocatalyze the one or more target gases.
 13. The product as recited inclaim 10, wherein the at least one biocatalyst being operable to converta C1-C3 hydrogen-comprising target gas to a product, wherein the C1-C3hydrogen-comprising target gas is selected from the group consisting of:methane, ethane, ethylene, propane, and propylene.
 14. The product asrecited in claim 10, wherein the microcapsules are arranged in apolymeric network in a configuration selected from the group consistingof: a microcapsule-embedded mesh; a packet containing the microcapsules;and a microcapsule-embedded adhesive.
 15. The product as recited inclaim 14, wherein the polymeric network comprises at least one polymermaterial and at least one inorganic material.
 16. The product as recitedin claim 14, wherein the polymeric network comprises at least a twophase polymer network.
 17. The product as recited in claim 14, whereinthe microcapsules are embedded in the polymeric network.
 18. The productas recited in claim 14, wherein the polymeric shell includes a blockpolymer/copolymer network.
 19. A product, comprising a plurality of themicrocapsules as recited in claim 1, wherein the microcapsules arearranged in a configuration selected from the group consisting of: apacked bed; a moving bed; a fluidized bed; a microcapsule-embedded mesh;a packet containing the microcapsules; and a microcapsule-embeddedadhesive.
 20. A microcapsule for selective catalysis of gases, themicrocapsule comprising: a polymeric shell permeable to one or moretarget gases; and at least one biocatalyst disposed in an interior ofthe polymeric shell, wherein the at least one biocatalyst comprises oneor more biocatalytic components selected from the group consisting of:one or more cell membrane fragments; one or more cytosolic cellcomponents; live cells; and reconstituted whole cells.
 21. Themicrocapsule as recited in claim 20, wherein the at least onebiocatalyst selectively oxidizes a C1-C3 hydrogen-comprising target gasto form a product.